Embodiments of the present invention relate generally to microparticles and more particularly, to microvessels that separate substances, such as biological or chemical substances, from an ambient environment.
Various protocols in biological or chemical research involve performing a large number of controlled chemical reactions within solutions or mixtures that are isolated from each other and/or from an ambient environment. Such isolated solutions or mixtures (i.e., reaction volumes) may be formed in assays by using test tubes, microcentrifuge tubes, and wells of microplates. For example, in multiplex assays, an unknown analyte having an identifiable label (e.g., fluorescent label) may be exposed to thousands of known probes under controlled conditions. Each known probe may be deposited into a corresponding well of a microplate. Observing any chemical reactions that occur between the known probes and the unknown analyte within the wells may help identify or reveal properties of the analyte. Generally, in assays such as the above, it is desirable to observe as many chemical reactions as possible in the least amount of time. It is also desirable to reduce costs and increase control and efficiency of the chemical reactions.
For example, a known quantitative PCR method uses a flat stainless steel plate that has two opposite plate surfaces and an array of through-holes extending completely through the plate between the plate surfaces. The through-holes are configured to hold nanoliter-sized reaction volumes of a liquid. The plate is chemically modified so that the plate surfaces are hydrophobic and interior surfaces of the through-holes are hydrophilic. The differential hydrophobic-hydrophilic quality retains liquid within the through-holes during the plate preparation process. Select primer pairs are inserted into through-holes in the plate so that each primer pair has a known through-hole location in the array. The primer pairs are immobilized onto the interior surfaces of the corresponding through-holes. Once the plate is prepared, a cDNA sample is mixed with fluorescent PCR reagents and loaded into the through-holes of the array. The through-holes are then sealed and the plate undergoes a thermal cycle pursuant to known PCR protocols. If a particular primer pair is capable of hybridizing with the cDNA sample, then mRNA having fluorescent properties will be amplified within the through-hole of that particular primer pair. Images of the plate are acquired and subsequently analyzed to determine which primer pairs amplified the mRNA and to what amount.
However, the above method may have certain challenges or limitations. For example, each primer pair must have a known through-hole location in order to identify the primer pairs that positively react with the cDNA sample. In other words, the reaction volumes within the through-holes are not separately identifiable, but must be identified by the through-hole's position in the array. Second, an imager or optical detector cannot detect amplification from a side of the through-hole but must face one of the plate surfaces in order to detect light emitting from the through-holes. As such, in assays that include real-time imaging or in assays that are interested in diffusion properties of the reactants, the image may provide limited information. Furthermore, the plate's size and shape limit or restrict the plate's use in systems where more sortable or transportable substrates are desired.
Another method that seeks to form separate reaction volumes is known as “emulsion PCR.” Emulsion PCR may be used to address problems where unwanted DNA fragments are amplified in conventional PCR amplification. In the emulsion PCR method, an oil-surfactant mixture is mixed with an aqueous solution to form tiny aqueous micelles that are separated from each other by the oil-surfactant mixture. The aqueous solution includes DNA fragments as well as other PCR components for amplifying the DNA fragments. A density of the DNA fragments compared to the rest of the aqueous solution is relatively small so that when the aqueous solution is mixed with the oil-surfactant mixture to make the aqueous micelles, there are at most a few DNA fragments in each aqueous micelle. The emulsion is then subjected to known PCR protocols to amplify the DNA fragment(s) in each aqueous micelle. Each aqueous micelle that contains at least one DNA fragment effectively functions as a bioreactor where the DNA fragment is amplified. With very few DNA fragments in the aqueous micelles, unwanted DNA fragments are not amplified.
One known pyrosequencing method uses emulsion PCR to sequence, for example, genomic DNA on a large number of capture beads. Each capture bead includes one sstDNA fragment (single-stranded DNA fragment) that is immobilized on the capture bead. The capture beads are added to a water-in-oil mixture similar to the emulsion described above. When the aqueous micelles are formed, each capture bead may be within one corresponding aqueous micelle. The aqueous micelles may then experience PCR thermal cycles to generate clonally amplified DNA fragments on the capture beads. After amplification, the capture beads are then added to corresponding wells in a microplate where each capture bead undergoes a sequencing-by-synthesis technique known as pyrosequencing. More specifically, nucleotides are sequentially delivered to the wells by flowing a solution containing a specific nucleotide through the microplate. When a nucleotide contacts a particular capture bead having template DNA strand with an appropriately complementary position, the nucleotide is added to a growing DNA strand that is hybridized to the template on the capture bead. Addition of a nucleotide that is complementary to the template DNA generates a fluorescent light signal that is captured by a CCD camera. The images are subsequently analyzed to determine the sequence of the genome.
However, emulsion PCR has limited applications. First, the aqueous micelles are difficult to individually identify and manipulate. Rather, information about the amplified nucleic acids within the aqueous micelles is typically determined through subsequent analysis and after the destruction of the aqueous micelles. Second, the aqueous micelles have limited sizes and shapes and have a limited stability since the surface tension properties are determined by the composition of the aqueous solution. As such, use of the emulsion PCR method is generally limited to situations when the composition of the aqueous solution forms stable aqueous micelles in the oil-surfactant mixture. Accordingly, emulsion PCR may not be suitable for assays that desire bioreactors having certain sizes or shapes. Third, after the aqueous micelles are formed in emulsion PCR, it may be difficult to manipulate or handle the aqueous micelles in a controlled manner. For example, it may be difficult to add reagents or other chemicals incrementally to the aqueous micelles. Furthermore, it may be difficult to add reagents or other chemicals selectively to certain aqueous micelles and not others.
Accordingly, there is a need for individually identifiable microvessels that separate reaction volumes from each other and/or an ambient environment. There is also a need for bioreactors that may be at least one of transported, sorted, and manipulated during a biological or chemical assay without destroying the reaction volume or somehow negatively affecting the chemical reaction therein. There is also a need for microvessels that may hold substances within reservoir cores where the substances and/or chemical reactions involving the substances may be detected externally. There is also a need for microvessels that store, transport, and release chemical substances in ways that they can be kept separated or combined for various steps of a synthetic or analytic process.